The present invention relates to biochemical diagnostic and assay methods and more specifically to the determine of very small quantities of chemical species involved in life processes, e.g., enzymes and enzyme substrates, antigens and antibodies, etc. This invention specifically relates to those analytical methods in which a chemical species to be determined, generally a bio-material, is coupled through intermediate reactions or reacts directly in an electromagnetic signal-generating system in which the species, or its progeny in the case of intermediate reactions, is converted into an end product with the concomitant release of electromagnetic radiation.
Life processes involve a staggering variety of biochemical reactions, all interrelated, and occurring either simultaneously, or in carefully regulated sequences. Many processes that may involve relatively massive amounts of materials, e.g. metabolic processes, may, in turn, be regulated by minute amounts of bio-materials, e.g. enzymes or hormones. In other instances, malfunctions and/or diseases of the organism may release extremely small amounts of biomaterials from their normal environments into other systems of the organism. The detection and quantification of these biomaterials, both in their normal environment and in abnormal environments can yield a great amount of information concerning the functioning of both major and minor systems in the organism, and can indicate system malfunction and/or disease, as well as invasion by foreign bodies such as bacteria or viruses. Such a bio-material is thus generally defined as any chemical compound found in living organisms.
In recent decades, various techniques have been developed for determining very small quantities of biomaterials. These techniques may utilize, for instance, radioactive tracer techniques, fluorometric techniques, colored dye development, bioluminescence, chemiluminescence, etc. Such techniques depend on inherent characteristics of the materials of interest that give rise to signals that can be detected on suitable instrumentation; or by combining or associating materials that generate, or can be induced to generate such signals, with the molecular species of interest.
The particular analytical technique to which this invention specifically relates involves electromagnetic radiation generating reactions, more particularly those electromagnetic radiation generating systems in which light is produced either by the reaction of a bio-material with a protein or by the enzyme-potentiated reaction of the material with a second chemical species. Such systmes derived from living systmes and which involve proteins, including enzymes, are defined herein as bioluminescent reactions. They have been extensively discussed in the literature. For example, see Johnson et al in Photophysiology, Vol. 7, pages 275-334 (1972). The sources of reagents for such reactions as well as purification techniques for the reagents are well known. By "electromagnetic radiation generating" applicant means chemical systems which emit electromagnetic radiation upon the reaction of the system components with one another, whether or not the reaction requiries a catalyst, whereby at least one product is yielded which was not a component of the unreacted system.
The electromagnetic radiation produced by bioluminescent reaction systems is, directly proportional to the amount of the reaction limiting component available for entering into the reaction. By way of illustration, light is produced when the enzyme, luciferase, acts to oxidize the substrate, fire-fly luciferin, in the presence of co-factor, adenosine triphosphate (ATP) and oxygen. The reaction may be summarized: ##EQU1## Where AMP is adenosine monophosphate, and P--P is disphosphate. The oxidation of each luciferin molecule yields a specific quantity of light, with the total light yield being directly proportional to the number of molecules oxidized. Thus, a measurement of the light yield indicates the number of luciferin, ATP, or oxygen molecules entering into the reaction, depending upon which of the three components is in molar excess, or the activity of the catalyst, luciferase.
If ATP is the least abundant species, then the reaction will cease when all the ATP is used up; or, if oxygen is the least abundnant, then when all the oxygen is used up. By the same token, the activity of the luciferase can be ascertained, if its activity is the limiting factor in the reaction process. The most accurate and complete results are obtained by ensuring a molar excess of all the components of the bioluminescent system other than the one of unknown concentration or activity to which the assay is directed.
Similar considerations apply in the use of the bacterial luciferase system for analysis of chemical species, e.g. bio-materials. Here, bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide with oxygen in the presence of a long carbon chain aldehyde to yield light, among other products. This system is typically employed to determine reduced flavin mononucleotide. The flabin mononucleotide may be the product of a reaction or series of reactions in which the flavin mononucleotide is eventually produced in a quantity that is directly proportional to a chemical species reacted at the first of the series. For example, dehydrogenases such as flavin mononucleotide oxidoreductase will oxidize the reduced from of nicotine adenine dinucleotide, which in turn may be produced by other dehydrogenases, to reduced flaving mononucleotide. The product flavin mononucleotide is then employed as the limiting component in the bacterial luciferase system. The light so generated is a measure of the original reduced nicotine adenine dinucleotide. A multiplicity of reactions of this nature may be coupled together to yield a product which is determinable by a bioluminescent reaction. As a consequence, any chemical species which can be reated to eventually yield a stiochiometrically equivalent quantity of ATP or reduced flavin mononucleotide may be assayed, respectively, by the fire-fly and bacterial luciferase systems. The prior art has employed the foregoing bioluminescent reactions in qualitative or quantitative coupled and direct assays. For example, see Hammerstedt, "Analytical Biochemistry" 52:449-455 (1973); Brolin et al., "Analytical Biochemistry" 39:441-453 (1971); and Mansberg, U.S. Pat. No. 3,679,312. In those cases where these assays have heretofore been conducted in a liquid environment all of the reagents were in solution and thus distributed homogeneously throughout.
When assaying for very low quantities of chemical species, or very low activities of enzymes, the quantity of electromagnetic radiation generated by processes such as noted above, is correspondingly small. In addition, since the reactions have heretofore been carried out in solution, the radiation emitting components are dilute and the radiation is emitted throughout a volume whereby the radiation intensity is lower than if high concentrations of reagents could be employed. This adversely affects the assay sensitivity. In addition, the larger and more opaque the volume of liquids, the greater is the possibility of self-absorption of the emitted radiation before it can leave the solution and be detected by suitable instrumentation. Finally, prior techniques measure the radiation as light emitted from a transparent container. However, irregularities in the container wall will scatter the light unpredictably, thus introducing variation into the assay.
Another detriment of conducting electromagnetic radiation assays in solution is the loss of costly reactants such as, for instance, enzymes and co-enzymes. Generally, there is no simple means of recovering such materials from solution, and they must, therefore, be discarded and replaced by new reactants for each successive assay.
In order to conserve costly enzyme materials and recover them for subsequent use, it has become well known in the art to immobilize various enzymes to insoluble support members or to one another so that the material is not lost or leached into solution during the reaction processes. See, for instance, U.S. Pat. Nos. 3,925,157 to Hamsher; 3,930,950 to Royer; 3,959,079 to Mareschi et al; 3,542,662 to Hicks et al, all of which describe various means and materials for attaching enzymes to support materials. H. H. Weetall has reviewed the chemistry of enzyme immobilization in "Analytical Chemistry" Volume 46, pages 602A et. seq., (1974) and the applications of immobilized enzymes has been discussed in "Analytical Chemistry," Volume 48, pages 544A et. seq. (1976). The prior art has, however, not disclosed immobilizing bioluminescent proteins such as the luciferases so that they can be recovered from the test solution and used over. Similarly, it is heretofore unknown to immobilize flavin mononucleotide oxidoreductase.